As the dome-shaped, transparent outermost part of the eye, the cornea provides the majority of the focusing power for the visual pathway. When damaged due to severe injury or disease, its normally smooth contour and optical clarity are often lost, resulting in reduced vision and, in many cases, blindness. Restoration of normal corneal structure and function in Service Members and Veterans with vision-compromising corneal injuries and disease is a priority. In spite of the various types of cadaveric corneal transplants that are available, there remains a major clinical need for new modalities to rapidly reconstruct and regenerate corneal tissue after severe injury or disease. We propose to develop a novel, sutureless, corneal tissue substitute that stabilizes deep ulcers, defects, and thinned areas of the cornea. The material is applied to a corneal wound as a viscous liquid, forming a crosslinked, transparent gel within minutes that recreates the smooth, air-cornea interface necessary for clear vision, while also promoting rapid epithelialization. This technology leverages a novel collagen crosslinking modality known as copper-free click chemistry that is bio-orthogonal: it does not react with proteins, cells, or biologic systems of any kind. As such, it can be safely applied to the surface of a wounded cornea without producing toxic side products, and without the need for potentially harmful external catalysts or triggers. We hypothesize that while a bio-orthogonally, in situ-crosslinked, collagen gel alone may promote rapid epithelialization and wound stabilization, its long-term biointegration and transparency will be enhanced by the presence of encapsulated corneal stromal stem cells that can re-model the applied matrix without causing fibrotic changes. We will test this hypothesis by evaluating the in vitro and in vivo performance of the collagen gel with and without encapsulated corneal stromal stem cells. Corneal stromal stem cells are known to secrete factors critical to preserving the transparency of the cornea if they are able to quiescently differentiate into keratocytes rather than myofibroblasts. In preliminary work, we have shown that bio-orthogonally crosslinked collagen gels are able to encapsulate corneal stromal stem cells and facilitate keratocytic morphology, and also support the formation of a multi-layered epithelium ex vivo. Motivated by this data, our first aim is to characterize and control the biological response of corneal tissue to in situ-crosslinked collagen gels in vitro. Our second aim is to evaluate the biointegration and functional performance of in situ- crosslinked collagen gels as a corneal stromal substitute in vivo. This research will increase understanding of a novel class of sutureless, in situ-forming tissue scaffolds to reconstruct corneal tissue. The results from these studies will build the foundational data for a VA Merit Award and future clinical translation of this technology for the benefit of Service Members and Veterans at risk of or suffering from corneal blindness.